Modified tachykinin receptors

ABSTRACT

The invention provides a modified tachykinin receptor in which the three amino acids of the DRY sequence that occurs adjacent to the junction of the TM3 domain with intracellular loop 2 are replaced with amino acids whose side chains are neither lipophilic nor contain charged groups. The receptor exhibits similar ligand binding characteristics to the wild type receptor but is incapable or substantially incapable of initiating an endogenous signal. Thus the ligand exhibits no or substantially no intra-cellular coupling of the receptor to the G protein, whereby there is substantially no transduction of ligand binding signals to the cell, The invention also includes fragments of the receptor containing the modified DRY sequence, and polynucleotides that encode the modified receptor as aforesaid. Therapeutic and diagnostic uses for the receptor are disclosed.

FIELD OF THE INVENTION

The present invention relates to a nucleic acid sequence encoding atachykinin receptor, the tachykinin receptor encoded by said sequence,methods for its preparation and its use in therapy and screening. Inparticular, the invention relates to a modification to tachykininreceptor proteins that gives rise to unexpected and useful properties.

BACKGROUND TO THE INVENTION

Tachykinins are important in the mediation of many physiological andpathological processes including inflammation, pain, migraine, headacheand allergy induced asthma. They belong to an evolutionary conservedfamily of peptide neurotransmitters that have an established role inneurotransmission. They share the C-terminal sequencePhe-Xaa-Gly-Leu-Met-NH₂ (SEQ ID NO 12) in which Xaa represents ahydrophobic residue. That sequence is characteristic of tachykinins andbelieved to be mainly responsible for their biological activity atneurokinin receptors.

Mammalian tachykinins include substance P (SP), neurokinin A (NKA) andneurokinin B (NKB) which exert their effects by binding to specificreceptors. SP is the most prominent member of the tachykinergic systemand is released from sensory nerve endings throughout the body. Itsamino acid sequence is:H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂   (SEQ ID N^(o)1)The amino acid sequence for NKA is:H-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH₂   (SEQ ID N^(o)2)The amino acid sequence for NKB is:H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH₂   (SEQ ID N^(o)3)

SP has been implicated in the pathology of numerous diseases. Forexample, it has been shown to be involved in the transmission of pain,in conditions associated with vasodilation, smooth muscle contraction,bronchoconstriction, activation of the immune system and neurogenicinflammation. It has been implicated in migraine, as well as indisorders of the central nervous system, such as anxiety andschizophrenia; in respiratory and inflammatory diseases, such as asthmaand rheumatoid arthritis; and in gastrointestinal (GI) disorders anddiseases of the GI tract, such as ulcerative colitis and Crohn'sdisease.

Tachykinin receptors include NK-1, NK-2 and NK-3 and are membraneproteins of the super-family of guanine nucleotide-binding protein(G-protein)-coupled receptors (GPCR). They have extra-cellular bindingsites that have preferential affinities for the ligands SP, NKA and NKBrespectively. Like other G-protein coupled receptors, they possess anextra-cellular N-terminus, an intra-cellular C-terminus and seventrans-membrane (TM) α-helices of 20-30 amino acids connected by first,second and third extra-cellular loops and by first, second and thirdintra-cellular or cytoplasmic loops. The greatest homology is found inthe membrane-spanning α-helices of trans-membrane domains TM1-TM7 whilethe N- and C-termini show greater diversity between the three types ofneurokinin receptor.

Cloned NK-1 receptors have been reported, including those for the Ranacatesbeina, (Simmons et al., Neuroscience, 79: 1219-1229 (1997)), Musmusculus, (Sundelin et al, Eur. J. Biochem. 203: 625-631 (1992)), Rattusnorvegicus, (Hershey et al, J. Biol Chem., 266: 4366-4374 (1991) andYokota et al, J. Biol. Chem., 264: 17649-17652 (1989)), Cavia porcellus,(Gorbulev et al., Biochem. Biophys. Acta 1131: 99-102 (1992)), and Homosapiens (human), (Takeda et al, Biochem. Biophys. Res. Comm. 179:1232-1240 (1991) and Fong et al U.S. Pat. No. 5,525,712 and U.S. Pat.No. 5,584,886). Cloned rat and bovine neurokinin-2 receptors have beenreported (Y. Sasi et al., Biochem. Biophys. Res. Comm., 165: 695 (1989),and Y. Masu, et al., Nature 329: 836 (1987)). Cloned rat neurokinin-3receptor has also been reported (R. Shigemoto, et al., J. Biol. Chem.,265:623 (1990)).

All three receptors share the signal transduction mechanisms of aG-protein coupled receptor. The receptor is in an OFF state when noligand is present, but, is triggered into an ON state when an agonistligand binds to the receptor. The G-protein when in its ON statetriggers a downstream signaling pathway or signal cascade. The G-proteincomprises α-, β- and γ-units that are bound together in the OFF state,the α-subunit then having GDP bound to it. In the ON state GTP replacesthe GDP bound to the α-subunit. The α-subunit becomes dissociated fromthe β- and γ-subunits and becomes available for activating the signalcascade. After a short period, the GTP becomes hydrolyzed to GDP and theG-protein returns to its non activated OFF state. Hydrolysis provides anegative feedback mechanism that ensures that the G protein is only inits activated ON state for a short period.

Various studies have been undertaken, involving different G-proteinreceptors, to determine how the various regions of the protein structureaffect intra-cellular coupling of the receptor to the G-protein andconsequential transduction of ligand binding signals to the cell.G-protein receptors have a well-conserved sequence in the secondintracellular loop where the loop joins the third trans-membrane domainthat is known as the DRY sequence. It has the residues5′-DRYXXV(P)XXPL-3′  (SEQ ID N^(o)4)in which L represents Leu, Ile, Val, Met or Phe and X represents anyamino acid. It has been suggested that the DRY sequence contributes tothe efficient binding and activation of G-proteins. Fraser et al., Proc.Natl Acad Sci. USA, 85: 5478-5482 (1988) report a change of Asp to Asnat position 130 of the human β-adrenergic receptor (i.e. the D of theDRY sequence) resulting in human β-adrenergic receptor that exhibitshigh affinity binding of agonist whilst being unable to interacteffectively with G-protein. However, a second paper from the samelaboratory reports that the previous very high agonist bindingefficiency in human β-adrenergic receptor mutated at position 130, uponwhich the above mentioned conclusion had been based, had not beenreproduced (Wang et al., Mol Pharmacol 40(2): 168-79 (1991)). In areview article Savarese and Fraser said that this locus may be importantfor coupling to some, but not all, G-proteins (Biochem J., 283: 1-19(1992)). Moro et al made mutants of the Hm1 muscarinic cholinergicreceptor with changes towards the 5′-end of the DRY sequence and foundthat replacing L at position 131 with A gave the strongest reduction incoupling efficiency (J. Biol. Chem. 268: 22273-22278 (1993)).Subsequently, Shibata et al made a mutant of the angiotensin II receptortype I in which DRY at positions 125-127 is replaced by GGA and M atposition 134 is replaced by A, resulting in uncoupling of the mutant Areceptor from G-proteins (Biochem. Biophys. Res. Com. 218: 383-389(1996)). The authors concluded that DRY sequence as a whole includingthe final lipophilic amino acid L serves as a general site for G-proteincoupling but they did not go on to consider what effects might beobtained by change confined to the DRY portion of the sequence.

Comparing the binding affinities of the two known isoforms of the humanNK-1 receptor shows the importance of the cytoplasmic tail. The longform (407 amino acids) and short form (311 amino acids) differ in thelength of the C-terminus. The long form has similar substance P bindingcharacteristics to the rat NK-1 receptor, while the short form of thereceptor has an apparent substance P binding affinity 10-fold less thanthe rat NK-1 receptor.

Furthermore, studies on these and other receptors have shown that theeffect of mutations is unpredictable and specific to certain families ofreceptors. Hence, a change in one G-protein coupling family will notnecessarily have the same effect on another. Other attempts todistinguish protein binding and signaling effects bear this out.

SUMMARY OF THE INVENTION

The invention provides a mutant tachykinin receptor in which the threeamino acids of the DRY sequence that occurs adjacent to the junction ofthe TM3 domain with intracellular loop 2 are replaced with amino acidswhose side chains are neither lipophilic nor contain charged groups,said receptor exhibiting similar ligand binding characteristics to thewild type receptor but being incapable or substantially incapable ofinitiating an endogenous signal. Thus the ligand exhibits no orsubstantially no intra-cellular coupling of the receptor to theG-protein, whereby there is substantially no transduction of ligandbinding signals to the cell. The way in which tachykinin receptorsattach to cell membranes, the trans-membrane domains extra-cellular andcytoplasmic loops and the place where the DRY sequence referred to aboveoccurs are apparent by inspection of FIG. 5 of the accompanyingdrawings.

The invention further provides any of the following:

-   -   a fragment of said mutant tachykinin receptor containing said        modified DRY sequence;    -   an isolated protein or polypeptide containing an amino acid        sequence at least 95% identical to the above sequence;    -   a variant thereof with sequential amino acid deletions from        either the C terminus or the N-terminus; and    -   an allelic variant, heterospecific homologue or biologically        active proteolytic or other fragment thereof containing said        modified DRY sequence.

The invention also comprises an isolated cell membrane in which amodified tachykinin receptor as aforesaid is incorporated as membraneprotein. Such cell membrane material finds utility for research and inparticular for screening for therapeutically useful compounds asdescribed below.

The invention yet further provides any of the following:

-   -   (a) an isolated nucleic acid molecule comprising a        polynucleotide that encodes a modified tachykinin receptor as        aforesaid;    -   (b) an isolated nucleic acid molecule comprising a sequence that        is hybridizable to the above sequence;    -   (c) a gene which is the result of extending the above sequence        or any sequence that is hybridizable to the above sequence;    -   (d) a sequence or gene that is functionally equivalent to the        above sequence or to a gene that is an extension of the above        sequence, i.e. that is not identical to the sequence or gene        referred to but functions biologically as equivalent to the        sequence or gene referred to, including any allelic variants and        heterospecific mammalian homologues, including artificial or        recombinant sequences created from cDNA or genomic DNA;    -   (e) a recombinant vector comprising the above gene sequence; and    -   (f) a host cell transformed with the vector.

The invention also provides a method for producing one of the amino acidsequences described herein and especially the receptor protein definedby SEQ ID N^(o)5, which method comprises the steps of:

-   -   (a) inserting said nucleic acid sequence into an appropriate        vector;    -   (b) culturing in a culture medium a host cell previously        transformed or transfected with the recombinant vector of step        (a);    -   (c) harvesting cells containing the receptor protein obtained        from step (b); and    -   (d) separating or purifying from said culture medium or from        said host cells the thus-produced receptor protein. In step (d)        of the above method, the receptor protein may be obtained either        from the culture medium and/or by lysing the host cell, for        example by sonication or osmotic shock.

The invention yet further provides a method for screening fortherapeutically active compounds, said method comprising the followingsteps:

-   -   (a) providing a cell line expressing a modified tachykinin        receptor as aforesaid;    -   (b) adding test sample to a solution containing labeled SP or        other tachykinin ligand and the cell line from step (a);    -   (c) incubating the cell line, test sample and labeled SP or        other ligand mixture from step (b) to allow binding of SP or        other ligand and test sample to the modified tachykinin        receptor;    -   (d) optionally separating the non-bound labeled SP or other        ligand from the labeled SP or other ligand bound to the modified        tachykinin receptor and, if desired,    -   (e) measuring the amount of labeled SP or other ligand that is        bound to the modified tachykinin receptor.

The invention further provides:

-   -   (a) the use of a modified tachykinin receptor according to the        invention in the preparation of a medicament for the treatment        or prophylaxis of a condition associated with substance P or        other tachykinin (neurokinin) receptor-binding ligand;    -   (b) the use of such a modified tachykinin receptor in therapy;    -   (c) a method for the treatment or prevention of a condition        associated with over-expression of an endogenous tachykinin        ligand, which method comprises administration to a patient in        need thereof of a non-toxic, effective amount of such a modified        tackykinin ligand as described above;    -   (d) a composition comprising a modified tachykinin ligand as        described above in association with a pharmaceutically and        pharmacologically acceptable carrier therefor; and    -   (e) a use, method or composition according to any one of (a)        to (d) above, in which the modified tachykinin ligand becomes        generated in vivo from a nucleic acid sequence encoding such a        ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described with reference to theaccompanying drawings, in which:

FIG. 1: describes a protein sequence, being a translation of thesequence shown in FIG. 1 and being the sequence of a modified human NK-1receptor according to the invention (hNK-1Rv1).

FIG. 2: is a consensus cDNA sequence encoding a modified human NK-1receptor according to the invention.

FIG. 3: is a bar chart showing the results of Calcium imagingexperiments on NK-1 wild type receptor and the modified receptor of FIG.1 (hNK-1Rv1), and wild type receptor antisense transfected COS cells.

FIG. 4: shows the sequence alignments between NK-1 and other NKreceptors in various species in the region of the DRY motif in TM3,demonstrating high conservancy (In the Figure, NK-1 sequences 1-4provide SEQ ID NO 13, NK-1 sequence 5 provides SEQ ID NO 14, NK-2sequences 1-2 and 4-7 provide SEQ ID NO 15, NK-2 sequence 3 provides SEQID NO 16 and NK-3 sequences 1-3 provide SEQ ID NO 17).

FIG. 5: is a diagram showing the nNK-1Rv1 receptor and portions of thecell membrane into which it is incorporated (SEQ ID NO 18; in additionto the DRY motif, L223 is changed to I; this change is believed to beinconsequential).

DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

Definitions for a number of terms used in this specification are givenbelow:

Alleles or allelic sequences mean alternative forms of the receptorgenes referred to above resulting from one or more variations in thenucleic acid sequence, resulting in altered mRNAs and proteins orpolypeptides

Functional equivalence when used in relation to gene sequences or aminoacid sequences covers sequences that are not identical to the sequencereferred to but function biologically or chemically as equivalents ofthe disclosed sequence.

Incapable of initiating an endogenous signal in the present contextmeans that, within experimental error (±5% of control), the modified NKRdoes not result in conversion of GTP to GDP in the cell or a recombinantconstruct; and/or

-   -   does not evoke a calcium flux therein; and/or    -   does not evoke other components of the signal transduction        pathway.

Isolated, when used in relation to a polynucleotide sequence, means sucha sequence that has been removed from its natural environment, i.e. fromthe organism in which it occurs in nature and/or from genes that areimmediately contiguous (one at the 5′ end and the other at the 3′ end)in the naturally occurring genome of the organism from which it isderived. Isolated when used in relation to a cell membrane refers to themembrane as a discrete entity substantially separate from cytoplasmicmaterial.

Operably linked refers to a linkage of polynucleotide elements in afunctional relationship. For instance, a promoter or an enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. More specifically, two DNA molecules (such as apolynucleotide containing a promoter region and a polynucleotideencoding a desired polypeptide or polynucleotide) are said to be“operably linked” if the nature of the linkage between the twopolynucleotides neither results in the introduction of a frame-shiftmutation nor interferes with the ability of the polynucleotidecontaining the promoter to direct the transcription of the codingpolynucleotide.

Stringent hybridisation conditions is a recognized term in the art andfor a given nucleic acid sequence refers to those conditions whichpermit hybridisation of that sequence to its complementary sequence andnot to a substantially different sequence. It generally implies at leastabout 97% identity between the sequences.

Protein and Nucleic Acid Sequences

In the above mentioned mutant receptors, amino acids for modifying theDRY sequence that are non-polar and have the correcthydrophilic/lipophilic balance include glycine and alanine which arepreferred. However, the inventors also envisage the use of other nonpolar amino acids.

In a preferred aspect, the invention provides a nucleic acid sequence asshown in FIG. 2 [SEQ ID N^(o)6], which encodes the consensus amino acidsequence for the modified hNK-1v or a sequence that hybridizes theretounder stringent hybridization conditions.

The invention also provides a preferred hNK-1v receptor sequence shownin FIG. 1 [SEQ ID N^(o)5].

The invention further provides a nucleic acid sequence encoding areceptor protein having at least 80%, preferably 90%, more preferably95%, and most preferably 98% amino acid identity with the hNK-1Rv1encoded by the nucleic acid sequence of SEQ ID N^(o)6, and which

-   -   encodes protein that can bind to SP, but cannot initiate its        endogenous signal, or    -   encodes a peptide fragment thereof according to sequence (d), or    -   encodes a sequence complementary thereto according to sequence        (e), as defined hereinabove.

The invention also provides a protein that:

-   -   (a) has at least 80%, preferably 90%, more preferably 95%, and        most preferably 98% amino acid identity with the hNK-1Rv1        protein having the amino acid sequence of SEQ ID N^(o)5; and    -   (b) can bind to SP or to a peptide fragment thereof, or to a        sequence complementary thereto, but cannot initiate its        endogenous signal to G protein.

The hNK-1Rv1 variant of the NK-1 receptor shown in SEQ ID N^(o)5 hassimilar ligand binding characteristics to the wild type receptor, but isdeficient in cell signaling capabilities, as demonstrated by the resultsof tests described in Examples 4 and 5. Some mammalian receptors mayexhibit overall homology with the hNK-1 receptor of less than 80% (forexample, rat NK-2 receptor has about 48% homology to rat NK-1 receptor),but are nevertheless included within the scope of this invention in viewof their conservancy in the intracellular DRY region of TM3.Accordingly, the present invention provides a polypeptide having as lowas 40% overall amino acid identity with the hNK-1Rv1 protein, but havingat least 75%, such as at least 80%, preferably 90%, more preferably 95%,and most preferably 98% amino acid identity with the TM3 intracellularloop of the bNK-1Rv1 protein in the vicinity of the DRY motif, andhaving the properties previously specified.

In view of the high level of conservancy demonstrated by other NKreceptors in the DRY region of TM3 intracellular loop, and also the highlevel of conservancy observed in that region between human and otherspecies, the present invention also provides a nucleotide sequence thatencodes a modified neurokinin receptor, wherein the modification is orincludes substitution of the DRY motif within the intracellular loop ofTM3, namely, Asp¹²⁹, Arg¹³⁰ and Tyr¹³¹, by Gly, Gly and Ala,respectively, for example wherein the receptor is a modified NK-2 ormodified NK-3 mammalian receptor.

Vectors

The invention further provides a vector (e.g. a plasmid or virus)comprising a nucleic acid encoding the modified tachykinin receptor,especially the hNK-1Rv1 receptor or any other modified hNK-1R of thisinvention.

A recombinant vector of the invention comprises an expression vectorcomprising a nucleic acid sequence encoding the modified NK-R,especially the hNK-1Rv1, polypeptide. One suitable vector for theexpression of a human variant NK-1 receptor of the invention is abaculovirus vector that can be propagated in insect cells and in insectcell-lines. Expression requires that appropriate signals are provided inthe vector, said signals including various regulatory elements such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. The regulatorysequences of the expression vectors are operably linked to the nucleicacid encoding the modified tachykinin receptor, especially the humanNK-1 variant receptor. Generally, recombinant expression vectors includeorigins of replication, selectable markers permitting transformation ofthe host cell, and a promoter derived from a highly expressed gene todirect transcription of a downstream structural sequence. Theheterologous structural sequence is assembled in an appropriate framewith the translation, initiation and termination sequences, andpreferably a leader sequence capable of directing sequences of thetranslated protein into the periplasmic space or the extra-cellularmedium. Where the vector is adapted for transfecting and expressingdesired sequences in eukaryotic host cells, preferred vectors comprisean origin of replication from the desired host, a suitable promoter andan enhancer, and also any necessary ribosome binding sites,polyadenylation site, transcriptional termination sequences, andoptionally 5′-flanking non-transcribed sequences.

Suitable promoter regions used in the expression vectors according tothe invention are chosen taking into account the host cell in which theheterologous nucleic acids have to be expressed. A suitable promoter maybe heterologous with respect to the nucleic acid for which it controlsthe expression, or may be endogenous to the native polynucleotidecontaining the coding sequence to be expressed. Additionally, thepromoter is generally heterologous with respect to the recombinantvector sequences within which the construct promoter/coding sequence hasbeen inserted.

A recombinant vector of the invention may be used to amplify apolynucleotide derived from the nucleic acid sequence encoding themodified NKR, especially the hNK-1Rv1 polypeptide that has been insertedin a suitable host cell, this polynucleotide being amplified every timethe recombinant vector replicates.

DNA sequences derived from the SV40 viral genome, for example SV40origin, early promoter, enhancer, and polyadenylation sites may be usedto provide the require non-transcribed genetic elements.

The suitable promoter regions used in the expression vectors accordingto the invention are chosen taking into account the host cell in whichthe heterologous nucleic acids are to be expressed. A suitable promotermay be heterologous with respect to the nucleic acid for which itcontrols the expression or alternatively it can be endogenous to thenative polynucleotide containing the coding sequence to be expressed.

Additionally, the promoter is generally heterologous with respect to therecombinant vector sequences within which the construct promoter/codingsequence has been inserted. Preferred bacterial promoters are the LacI,LacZ, T3 or T7 bacteriophage RNA polymerase promoters, the lambda PR, PLand trp promoters (EP 0 036 776), the polyhedrin promotor, or the p10protein promoter from baculovirus (Kit Novagen; Smith et al., 1983);O'Reilly et al., 1992, Baculovirus expression vectors: A LaboratoryManual. W.H. Freeman and Co., New York).

Preferred selectable marker genes contained in the expressionrecombinant vectors of the invention for selection of transformed hostcells are preferably dihydrofolate reductase or neomycin resistance foreukaryotic cell cultures, TRP1 for S. cerevisiae or tetracyclin,rifampicin or ampicillin resistance in E. coli, or Levan saccharase formycobacteria, this latter marker being a negative selection marker.

Preferred bacterial vectors of the invention are listed hereafter asillustrative but not limitative examples:

-   -   pQE70, pQE60, pQE-9 (Qiagen), pD10, fephagescript, psiX174,        p.Bluescript SK, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene);        pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT,        pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL        (Pharmacia); pQE-30 (Qiagen).

Preferred bacteriophage recombinant vectors of the invention are P1bacteriophage vectors such as described by Sternberg N. L. (1992;1994).

A suitable vector for the expression of hNK-1vRa polypeptide of theinvention or a fragment thereof, is a baculovirus vector that can bepropagated in insect cells and in insect cell-lines. A specific suitablehost vector system is the pVL 1392/1393 baculovirus transfer vector(Pharmingen) that is used to transfect the SF9 cell line (ATCC N^(o)CRL1711) which is derived from spodoptera frugiperda.

The recombinant expression vectors of the invention may also be derivedfrom an adenovirus such as those described by Feldman and Steg. (1996)or Ohno et al. (1994).

Another preferred recombinant adenovirus according to this specificembodiment of the present invention is the human adenovirus type two orfive (Ad 2 or Ad 5) or an adenovirus of animal origin (French PatentApplication n^(o)FR 93 05 954).

Particularly preferred retroviruses for the preparation or constructionof retroviral in vitro or in vivo gene delivery vehicles of the presentinvention include retroviruses selected from the group consisting ofMink-Cell Focus Inducing Virus, murine sarcoma virus, and Ross SarcomaVirus. Other preferred retroviral 5 vectors are those described in Rothet al. (1996), in PCT Application WO 93/25 234, in PCT Application WO94/06920, and also in Roux et al. (1989), Julan et al. (1992) and Nadaet al. (1991).

Yet, another viral vector system that is contemplated by the inventionconsists in the adeno associated virus (AAV) such as those described byFlotte et al. (1992), Samulski et al. (1989) and McLaughlin et al.(1996).

Expression Systems

For expression of the modified neurokinin receptors, the inventionprovides host cells transformed (prokaryotic cells) or transfected(eukaryotic cells) with such a vector; and any cell, or live organism,including a non-human mammal, that has been genetically engineered toproduce such a polypeptide, said cell or live organism incorporatingexpressibly therein a nucleic acid sequence according to this invention.

Heterologous expression systems may be used to express cloned NK-1receptor, NK-2 receptor and NK-3 receptor, including human and non-humanmammalian variants thereof. The choice of expression system depends on anumber of factors including stability of protein expression, posttranslational modification and required yield. However, as a generalrule, the more complex the organism the lower the yield of expressedreceptor, but the greater the likelihood that the receptor will be inits native conformation. Several expression hosts are commonly available

Prokaryotic host cells, e.g. Escherichia coli DH5-α (see Sambrook etal., for a comprehensive guide to gene expression in E. coli).

-   -   Yeasts, e.g. Pichia pastoris. The feasibility of expressing NK-1        receptor in yeast has been demonstrated by Arkinstall et al        (1995, FEBS 275 183-187) who successfully expressed therein the        closely related receptor, human NK-2. Large-scale production of        a human G-protein coupled receptor in yeast was demonstrated by        Sizemann et al (1996, Receptors and Channels, Vol 4, 197-203).        In brief, a protocol to express a functional NK-1 receptor in        yeast can be represented by: (1) splicing the NK-1 cDNA into a        yeast expression vector; (2) Transforming this vector into        yeast; and (3) Selecting for yeast containing the NK-1 cDNA and        expression of the NK-1 cDNA.    -   Eukaryotic host cells, e.g. insect cells, non-mammalian        vertebrate cells and mammalian cells. Non-mammalian vertebrate        cell lines include Xenopus (frog) oocytes. Other cell lines        which are contemplated in this invention include HeLa cells        (ATCC N^(o)CCL2; N^(o)CCL2.1; N^(o)CCL2.2), Cv 1 cells (ATCC        N^(o)CCL70), COS cells (ATCC N^(o)CRL 1650; N^(o)CRL 1651), Sf-9        cells (ATCC N^(o)CRL 1711), C127 cells (ATCC N^(o)CRL-1804), 3T3        cells (ATCC N^(o)CRL-6361), CHO cells (ATCC N^(o)CCL-61), human        kidney 293 cells (ATCC N^(o) 45504; N^(o)CRL-1573) and BHK        (ECACC N^(o)84100 501; N^(o)84111301); PC12 (ATCC N^(o)        CRL-1721), NT2, SHSY5Y (ATCC N^(o) CRL-2266), NG108 (ECACC        N^(o)88112302) and F11, SK—N—SH (ATCC N^(o) CRL-HTB-11),        SK—N—BE(2) (ATCC N^(o) CRL-2271), IMR-32 (ATCC N^(o) CCL-127). A        preferred system to which the gene of the invention can be        expressed are cell lines such as COS cells, 3T3 cells, HeLa        cells, 292 cells and CHO cells. A preferred system for the        efficient expression of hNK-1vR involves the use of CHO and COS        cell lines. The gene can be expressed through an endogenous        promoter of native CHO or COS, or through an exogenous promoter.        Suitable exogenous promoters include such as SV40 and CMV, or        perhaps a eucaryotic promoter such as the tetracycline promoter.        The preferred promoter being CMV.

In some instances, it may be required to tag e.g. a human NK-1 variantreceptor prior to purification. The tag is then, in most instances,encoded into the nucleotide sequence that is needed to express thepolypeptide. Examples of such tags include, but are not limited tosequences encoding C-myc, FLAG, a sequence of histidine residues,haemaglutin A, V5, Xpress or GST. Most of these tags can be incorporateddirectly into the sequence, for instance through PCR amplification byincorporating the appropriate coding sequence in one of the PCRamplification primers. However, the tag can also be introduced by othermeans, such as by covalent binding of the appropriate nucleic acidsequence encoding the tag moiety, such as GST, with the 3′ or 5′ end ofthe nucleic acid sequence encoding the polypeptide sequence.Purification of the NK variant receptor may, in the case of the use of ahistidine tag, then be carried out by passage onto a nickel or copperaffinity chromatography column, such as a Ni NTA column. The polypeptidethus produced may optionally be further characterized, for example bybinding onto an immuno-affinity chromatography column on whichpolyclonal or monoclonal antibodies directed to the NK variant receptorhave been previously immobilised.

For some purposes it may be useful to provide mammals e.g. mice, rats orguinea-pigs in which in which modified human or non-human mammaliantackykinin receptors, e.g. NK-1v receptors, are present. Transgenicrats, mice and other mammalian cells may be produced by generating atargeting vector and transfecting the cells to be cultured e.g. usingthe gene targeting services of, e.g. DNX Transgenic Sciences. In thecase of transgenic mammals, stem cells are transfected, cultured toblastocytes and introduced into the uterus of a female mammal. Becausethey have in their cell membranes tachykinin receptors that bind toendogenous ligands such as SP but are incapable, or substantiallyincapable, of initiating their endogenous signal, the animals areuseful, in research into the effects of these ligands and thedevelopments of transduction inhibitors, especially because they canindicate the behaviour of the animal the presence of endogenous ligandbut in the absence of a transduced signal and in the absence ofantagonist, and can therefore provide a true control. (Silver, Lee M.Title: Mouse genetics—concepts and applications Publisher: New York;Oxford University Press 1995); P De-Felipe-C et al;Nature. 1998 Mar. 26;392(6674): 394-7).

Assays

The present invention further provides a modified tachykinin receptor,such as a human NK-1v receptor, used as a substitute in an assay toidentify and evaluate entities that bind to the wild type tachykininreceptor.

The invention also includes human NK-1v receptor used as a substitute inan assay in order to determine the concentration of substance P in bodyfluids in patients with arthritis, pain, migraine, anxiety,schizophrenia, asthma, rheumatoid arthritis, and in gastrointestinaldisorders and diseases of the GI tract, like ulcerative colitis andCrohn's disease.

As previously mentioned three tachykinin receptors have been identified,referred to as NK-1, NK-2 and NK-3 and they have respective endogenouslignads SP, NKA and NKB. Although each tachykinin has a preferredligand, each receptor has the ability to interact with the othertachykinin ligands, and the pathology of cross-ligand binding in diseasestates is poorly understood. Study of a system where NK-1, NK-2 or NK-3receptor response either singularly or in tandem has been diminished invitro or in vivo could significantly aid understanding of the role oftachykinin receptors and their ligands in disease states.

Screening Methods

The invention also provides a method for screening for therapeuticallyactive compounds, said method comprising the following steps:

-   -   (a) providing a cell line expressing the modified tachykinin        receptor, e.g. the human NK-1v receptor;    -   (b) adding test sample to a solution containing labeled SP or        other ligand and the cell line from step (a);    -   (c) incubating the cell line, test sample and labeled SP or        other ligand mixture from step (b) to allow binding of SP or        other ligand and test sample to the modified tachykinin        receptor;    -   (d) optionally separate the non-bound labeled SP or other ligand        from the labeled SP or other ligand bound to the modified        tachykinin receptor; and, if desired,    -   (e) measuring the amount of labeled SP or other ligand that is        bound to the modified tachykinin receptor.

Preferably, the assay involves COS-7 cell lines. Cell membranescontaining the modified tachykinin receptor, e.g. the human NK-1vreceptor, can be used instead of whole cells.

The SP or other tachykinin ligand e.g. NKA or NKB may be labeled by anymethod known in the screening art, e.g. by a radioactive label, such as¹²⁵I, or by a fluorescent label. In certain circumstances, (e.g.fluorescence polarization assays), bound and unbound SP or othertachykinin ligand do not have to be separated to quantify the amount ofSP or other ligand bound to the receptor. Alternatively, the SP or otherligand may be bound to a matrix, and labelled cells may be used toquantify the binding of a modified tachykinin receptor, such as humanNK-1 receptor, to the SP. In this case, the assay procedure may followthe steps set out below:

-   -   (a) a cell line is provided that expresses a modified tachykinin        receptor, such as the human NK-1v receptor;    -   (b) the cell line is labeled;    -   (c) the test sample and labeled cells are added to a matrix        binding SP or other ligand;    -   (d) the labeled cells, test sample and matrix-bound SP or other        ligand are incubated to allow binding of SP or other ligand and        test sample to the expressed modified tachykinin receptor;    -   (e) the labeled non-bound cells are separated from the SP or        other ligand bound cells; and, if desired,    -   (f) the amount of labeled cells containing the modified        tachykinin receptor, such as the human NK-1v receptor, that has        bound to SP or other ligand is measured.

In order to provide transgenic animals or cell lines for use in assays,which animals and/or cell lines comprise a sequence as described herein,general methods are known and may be adapted accordingly. Differenttypes of vectors including modified retroviruses, adenovirus,adeno-associated virus, herpes virus and plasmid DNA have been proposedas vehicles to introduce foreign genetic material into cells or tissues.

Protein Therapy

The invention also encompasses modified tachykinin receptors, especiallyhuman NK-1v receptor, for use in protein therapy to reduce the effectsof, or an excess of endogenous ligand.

Protein therapy can be used for the suppression of the action of SP ininterstitial fluid of the lungs. For example, a purified preparation ofa modified tachykinin receptor, such as human NK-1v receptor, (eg aliquid or powder carrier formulation) can be directly administered tothe airways. Once in the airways, the tachykinin variant receptor caninteract and bind to, for example, SP molecules. This has the effectreducing the amount of SP available for the endogenous NK receptor.Likewise, a modified tachykinin receptor, such as the human NK-1vreceptor, can be directly introduced to body cavities such as joints andinterstitial lung space where SP is present. The variant receptor hasthe capability to bind SP, therefor reducing the amount of SP availableto interact with the wild type receptor and causing down-regulation ofthe SP cellular response.

The invention provides a modified NK receptor, such as a human NK-1vreceptor, for use in removing or suppressing SP in body fluids, e.g. theinterstitial fluid of the lungs and fluid in the cavities of joints. Apurified preparation of a modified NK receptor, such as human NK-1vreceptor, (e.g. a liquid carrier formulation) may be administereddirectly to a joint. Once in the fluid- filled joint cavity, the NKvariant receptor interacts and binds to SP molecules thereby reducingthe amount of SP available to activate the endogenous NK receptor.

The hNK-1v receptor can reduce the effect of excess or inappropriatelyexpressed SP in patients with pain associated with migraine, neuralgia,diabetic, peripheral, AIDS-related and chemotherapy-induced neuropathy,and neuropathies of diverse origin; anxiety and anxiety disorders, suchas panic disorder, phobias and obsessive-compulsive behavior;schizophrenia; asthma; rheumatoid arthritis; and in gastrointestinaldisorders and diseases of the GI tract, for example ulcerative colitisand Crohn's disease. Other conditions or disease states that can betreated, ameliorated or prevented include: psychosomatic andpsycho-immunological disorders; attention deficit disorder;pre-menstrual (PMT or PMS) or late luteal phase syndrome; mania orhypomania; aggressive behavior disorders; emesis, including motionsickness, migraine-induced sickness and that arising from chemotherapy;postherpetic neuralgia; depression; inflammation; eating disorders, suchas obesity, bulimia nervosa and compulsive eating disorders; cognitivedisorders, such as dementia and amnestic disorders; movement disorders,such as dyskinesias, akinetic-rigid syndromes, Gilles de la Tourettesyndrome, tremor, or dystonia; schizophrenic disorders; substance abusedisorders; bipolar disorder; sexual dysfunction, including impotence;stress; alteration of circadian rhythmicity; Alzheimer's disease;bladder disorders; hypertension; angina; ischaemia; multiple sclerosis;chronic obstructive lung disease; scleroderma; CNS disorders; and otherconditions where excess tachykinin peptides such as SP are involved.Furthermore, the inventors believe that modification to the DRY motif ofNK-2 receptor and NK-3 receptor in a similar manner to that describedfor the NK-1 receptor above, would allow for the production of modifiedtachykinin receptors which could be used to remove their specificligands from body fluids.

Several factors need to be taken into account in protein therapies.These include: solublisation, maintenance of activity and stability ofthe protein, delivery and dose. The first three points are closelyrelated. Proteins are large relative to conventional drugs and theirbiological activity is dependent on their primary, secondary, tertiaryand in some instances quaternary structure being maintained. They oftenhave labile bonds and numerous chemically reactive groups in their sidechains. Disruptions of their structure by denaturation or aggregationcan lead to loss of activity or increase in immunogenicity. One of thekey problems in devising effective formulations for biologically activeproteins is to find a formulation that is both stable and biologicallyactive. The route of administration of the protein also plays asignificant role in formulation. Microsphere formulations can be usedfor injection, aid in the maintenance of stability and activity of theprotein, and offer the possibility of slow release formulations. Solidlarge powder formulations are most suitable for use in aerosol andtopical treatments.

Putney and Burke (Nature Biotechnology 1998: 153-157) outline variousmethods for producing microspheres. One such method is theatomization-freezing process. In this encapsulation method, themicronised solid protein is suspended in biodegradable polymers ofDL-lactic co-glycolic acid (PLGA) solution that is then atomised usingsonication or air-atomisation. This produces droplets that are thenfrozen in liquid nitrogen. Addition of ethanol at <−40° C., in whichboth the protein and the PLGA are insoluble, extracts the organicsolvent from the micro-spheres. This process is further described inU.S. Pat. No. 5,019,400. An alternative polymer for use in the processis methylene chloride polymer,to encapsulate growth hormone see Johnsonet al, Nature Medicine 2: 795-799 (1996). The following method can beused to incorporate modified NKR, such as human NK-1v receptor,post-purification into microspheres:

-   -   (a) concentrate the purified, active microsphere modified NKR,        such as human NK-1v receptor to >100 mg/ml in the presence of        stabilisers;    -   (b) add PLGA or methylene chloride polymer solution and mix;        then    -   (c) atomise the frozen suspension in liquid nitrogen to fix the        microspheres and extract with ethanol. Microspheres thus        produced should have a diameter in the μM order.

The invention further provides a method for treatment of a patient inneed thereof, which comprises administering to said patient acomposition in the form of an aerosol that comprises a modifiedtachykinin receptor as described above, e.g. an hNK-1Rv receptor.

SP or other ligand such as NKA or NKB can also be removed directly frombiological fluids using the modified tachykinin receptors describedabove. For example, purified human NK-1v receptor could be bound to asuitable matrix. Biological fluids containing SP can be passed over thematrix, so that the SP preferentially binds to matrix while othercomponents of the fluid do not and SP becomes preferentially removedfrom the biological fluid.

Nucleic Acid Therapy

The invention further provides a method for gene therapy treatment of apatient in need thereof, which comprises administering to said patient anucleic acid sequence, virus or plasmid encoding a modified tachykininreceptor as described above, e.g. an hNK-1Rv receptor. Different typesof vector including modified retroviruses, adenovirus, adeno-associatedvirus, herpes virus and plasmid DNA have been proposed as vehicles forintroducing foreign genetic material into the cells or tissues ofpatients, and can in principle be used to introduce the modifiedtachykinin receptor nucleic acid sequences referred to above. Theappropriate strategy for administering the nucleic acid sequence dependson the target tissue, disease state and longevity of the proposedtherapy.

Furthermore, down regulation of the effect SP on a cellular system couldbe achieved by expressing the hNK-1v receptor in cells other than thoseexpressing native NK-1. The expression of SP in these cells (which arein the proximity of cells expressing native NK-1 receptor) could havethe effect of “moping” up SP and reducing the available SP for thenative receptor to interact with.

Introduction of a modified NK receptor, such as a human NK-1v receptor,(by way of direct introduction of protein or introduction of anexpressible gene encoding this protein) into the outer membrane of cellsexpressing the wild type receptor gives rise to competition between thewild type and variant receptors for available SP. The variant receptorcompetes with wild type receptor for binding of available SP anddecreases the amount of SP available for the wild type receptor. As theNK-1v receptor is unable to transduce a signal upon SP binding, theresult is a down-regulation of the action of the wild type receptor.

In the treatment of lung tissue, researchers at Stanford UniversityMedical Center, California have initiated a trial of gene therapy forcystic fibrosis in which the active material is delivered to the lungsby aerosol. The active material consists of a version of the cysticfibrosis trans-membrane conductance regulator gene packed into anadeno-associated virus (AAV) shell. A similar route can be adopted fordelivering the hNK-1v receptor to the lungs of patients where theactivation of native hNK-1 receptors by receptor ligands requires to bedown-regulated. A strategy for down-regulating the action between SP andhuman NK-1v receptor in lung tissue can be adopted which is similar tothe strategy proposed for gene therapy in cystic fibrosis. The sametechnique of packaging genes into AAV and infection of the lung tissuewith aerosol AAV can be used to introduce hNK-1v. Samulski et al(University of North Carolina, Chapel Hill, N.C. 27599, USA) market avector which can be used to package human NK-1v receptor gene into AAV.

Alternatively, a plasmid containing the human NK-1v receptor (pCMVNK-1v)may be used directly in gene therapy. The plasmid may be prepared as alipid:DNA complex and administered to the lungs as an aerosol, seePillai et al., Pharm-Res 15(11): 1743-7 (1998), McDonald et al.,Pharm-Res 1998 15(5): 671-9(1998). This strategy can also be used withan NK-1 receptor promoter. A pre-requisite for the use of gene therapyfor humans is the ability to make sufficient pharmaceutical gradeplasmid DNA. This problem has been addressed by Prazeres et al inTIBTECH 17: 169-74 (1999).

Both rAAV and lipid:DNA complex may be administered to the lungs vianebulisers, e.g. airjet nebulisers. Methods for administration maygenerally follow the teachings of McDonald et al, Pharm Res 15(5): 671-9(1998); Yonemitsu et al, Gene Therapy 4(7): 631-8 (1997); McDonald etal, Human Gene Therapy, 8(4): 411-22 (1997); Bellon et al, Human GeneTherapy 8(1): 15-25 (1997); and Niven, Critical Review of TherapeuticDrug Carrier Systems 12(2-3): 151-231 (1995).

Both viral and plasmid therapeutic compositions can also be delivered toother target sites such as joints and nervous tissue by infusion andinjection.

EXAMPLES

Embodiments of the invention will now be described in the followingExamples

Example 1 Preparation of a Modified Human NK-1 Receptor (NK-1v)

The starting material used was a plasmid pRc/CMV (Invitrogen Co)containing a cDNA clone encoding the human NK-1 receptor. The cloneencoded 407 amino acids and was flanked by a Nco I site at the 5′ end(around the ATG start codon) and a Xba I site at the 3′ end followingthe stop codon. Human NK-1v receptor was made from the above plasmidusing a PCR-based strategy that mutated the DRY motif (Asp¹²⁹, Arg¹³⁰,Tyr¹³¹) at the end of third transmembrane helix to GGA.

Portions of the cDNA clone in the above plasmid were amplified in twoseparate PCR reactions using Pfu DNA polymerase (Stratagene), 20 cyclesof PCR using 1 min at 94° C., 1 min at 55° C., 4 min at 72° C., followedby 10 mins at 72° C., 50 pmol of each primer, 50 ng of plasmid DNA, 100μl reaction volume.

The first PCR reaction used the following primers to produce a predictedproduct of 473 bp: (SEQ ID N^(o)7) 5′ primer, sense-A: 5′-AAC TAG AGAACC CAC TGC TTA-3′ (SEQ ID N^(o)8) 3′ primer, anti-sense-A: 5′-GCC ATAGCG CCG CCA AAA GCC ACA GCC GT-3′

The second PCR reaction used the following primers to produce apredicted product of 888 bp: (SEQ ID N^(o)9) 5′ primer, sense-B: 5′-TTTGGC GGC GCT ATG GCT ATC ATA CAT CC-3′ (SEQ ID N^(o)10) 3′ primer,anti-sense-B: 5′-AGC TCT AGC ATT TAG GTG ACA-3′.

Primers anti-sense-A and sense-B contained 17 bases of overlappingcomplementary sequence at the 5′ ends of each to allow annealing of thetwo products to form the full length mutated receptor. All the aboveprimers were custom synthesized by Perkin-Elmer.

The two PCR products were gel-purified (QIAEX, Qiagen) and 50 ng of eachpurified product was added to a 100 μl Pfu PCR reaction and three roundsof PCR performed without primers to allow the two overlapping regions ofthe PCR products to anneal and extend. 50 ng of the flanking primers(sense-A and anti-sense-B) were added and 20 cycles of conventional PCRperformed. The resulting full-length product was purified, cloned intopBluescript (Stratagene) and fully sequenced to confirm the mutationshad been successfully made.

The clone was modified by site-directed mutagenesis using a ClontechTransformer kit to remove an internal Nco I restriction site using theoligonucleotide sense-C also from Perkin-Elmer. Sense C: 5′-CGC GGA GGCTTC TAT GGC TGC AT-3′ (SEQ ID N^(o)11)

The resulting cDNA was excised from the parental vector, spliced into amammalian expression vector, pCMV3.1 (Invitrogen). This cDNA was in anorientation which allowed expression of the hNK-1vR polypeptide. Stockplasmid was prepared by amplification in E. coli and the plasmid DNAsubsequently purified using a Qiagen endonuclease free DNA preparationkit, and the fidelity of the construct confirmed by DNA sequencing. Thenucleic acid sequence of the cDNA is SEQ ID N^(o)6 (FIG. 2) and thetranslated protein sequence is SEQ ID N^(o)5 (FIG. 1). Similarly control(negative control) constructs were prepared whereby wild type andvariant HNK-1R cDNA was spliced into the vector in an oppositeconfiguration to that described above. A further control (positivecontrol) was prepared by ligation of the parental wild type hNK-1R cDNAinto the vector in a manner which would allow hNK-1R polypeptide to beexpressed.

Example 2 COS-7 Cells that Transiently Express Human NK-1v Receptor

COS-7 cells were grown in DMEM culture medium supplemented with 10%foetal calf serum and 2mM glutamine and maintained under an atmosphereof 5% CO₂. Cells were passaged at approximately 70% confluence byreseeding to a concentration of approximately 10% confluence per 175 cm²flask. COS-7 cells were harvested and prepared for electroporation inEquibio electroporation buffer. Cells (5×10⁶ cells) were electroporatedat room temperature in a 4 mm gap cuvette in a final volume of 800 μlcontaining 30 μg of transforming plasmid DNA, with 250 volts, 1500 μF atinfinite resistance using an Equibio EasyJect plus electroporator.Transformed COS-7 cells were cultured for 2-3 days in a 175cm² flaskprior to assay.

Alternatively cells (approximately 5-50 ul of transfected cells) werecultured in 6 well culture dishes containing 22mm diameter cover slips.These cover slips coated in cells were used for imaging experimentsdesigned to investigate changes in the concentration of intracellularfree calcium ([Ca²⁺]i). Cover slips were prepared by first immersing thecover slips in approximately 70% ethanol and then quickly passingthrough a gas flame to sterilize. Cover slips were then allowed to airdry in a culture hood prior to placing in the bottom of a culture wellof a standard six well culture dish. Cells were then cultured asdescribed above.

Example 3 COS-7 Cell Membranes Containing Human NK-1v Receptors

The transfected COS-7 cells of Example 2 were harvested by treatmentwith Versene. (Gibco BRL). Versene was used, as it allows detachment ofthe cells from the flask without substantial perturbation to cellmembrane proteins, then, the cells were washed once by re-suspending inassay buffer (50 mM Tris HCl pH 7.4, 3 mM MnCl₂, 0.02% BSA, 40 μg/mlbacitracin, 2 μg/ml chymostatin, 2 μM phosphoramidon, 4 μg/ml leupeptin)and centrifuged at 1000 g for 5 min. Cells were re-suspended in 5 ml ofthe assay buffer and a cell count performed prior to lysing cells, usinga Brinkman polytron at setting 6 for 15 seconds. The homogenate wascentrifuged at 20000 g for 10 minutes. Membrane pellets werere-suspended in assay buffer and stored frozen as 0.5-1 ml aliquotsuntil required for use.

Example 4 Ligand Binding to Receptors in Cell Membranes

COS-7 cells were transiently transfected with pCMV3.1 (Invitrogen)containing either parental wild type human NK-1 receptor cDNA or thevariant human NK-1 receptor cDNA of Example 1, and cell membranes wereprepared 2-3 days post-transfection using the procedure of Example 3.Radioligand binding studies were performed using [¹²⁵I]BH substance P tolabel NK-1 receptors.

Non-specific binding was defined by the NK-1 receptor-selective agonist[Sar⁹, Met(O₂)¹¹]substance P (Bachem) at a final concentration of 1 μM.On the day when they were required, the membrane suspensions were thawedand diluted as appropriate with assay buffer and incubated with varyingconcentrations of [¹²⁵I]Bolton-Hunter Substance P (0.05-3 nM, fromAmersham Life Sciences) for 50 minutes at 21° C. Saturation analyseswere performed to determine the affinity constants and maximum bindingcapacity for each receptor, by incubating membranes with increasingconcentrations of the radioligand, in the presence and absence of [Sar⁹,Met(O₂)¹¹]substance P. Reactions were terminated by rapid filtrationunder vacuum, onto GFC filters pre-soaked with 0.2% polyethylenimine.

[¹²⁵I]BH substance P bound with high affinity to both wild type andmutant receptors (Kd values of 2.218±1.107 nM (n=3) and 1.446 nM (n=2)respectively). Unpredictably, there appeared to be no significantdifference between the dissociation constant and the maximum bindingcapacity of the wild type and variant NK-1 receptors, indicating thatthe mutation does not affect agonist binding at the NK-1 receptor. Themaximum binding capacity can be directly compared, as shown in Table 1:TABLE 1 Wild Type Variant HNK-1 (n = 3) hNK-1 (n = 2) Kd (nM) 2.218 + −1.1107 1.446 Bmax 2.201 3.000

Example 5 Analysis of Intra-Cell Receptor Coupling in COS-7 Cells byChanges in Intra-Cellular Free Calcium Concentration

Coverslips containing cells were prepared and maintained as described.Cultured cells were washed twice in a Krebs-Hepes extra-cellular mediumbuffer (EM, composition in mM: NaCl 118, KCl 4.7, MgSO₄ 1.2, CaCl₂ 1.2,KH₂PO₄ 1.2, Hepes 10, glucose 11 and BSA 0.1%, (pH 7.2 at 20° C.,(Rossant, et al Endocrinology, 140, 1525-1536), and then loaded withFura-2 (Rossant, et al Endocrinology, 140, 1525-1536) by incubation for3 h at 20° C. with EM containing Fura-2-AM (2 μM, Molecular Probes).This procedure enables the cells to load with Fura-2-AM, which becomeshydrolysed to the free acid form once inside the intact cells. Afterloading, coverslips were mounted into imaging chambers and perfused withEM to remove extra-cellular Fura-2-AM and to allow hydrolysis ofintracellular Fura-2-AM to occur. Measurements of changes in the free[Ca²⁺]_(i) in individual cells were made from the fluorescence ratio(excitations 340 nm/380 nm, emission >510 nm) using a Spectral Wizardmonochromator, cooled integrating CCD camera and a dedicated suite ofsoftware (Merlin, Life Sciences Resources, Cambridge, UK). Data areexpressed as ratio units 340/380.

Genes encoding wild type sense, wild type anti-sense and variant NK-1receptors described above were transiently transfected into COS-7(monkey kidney) cell lines. Two to three days post-transfection, cellswere loaded with FURA-2-AM dye (whose fluorescence is [Ca²⁺]_(i)dependent) and challenged with substance P. The [Ca2+]_(i) was followedby monitoring the change in light emitted (>510 nm) when excitationlight of either 340 nm or 380 nm was sequentially used to illuminate theloaded cells and expressed as a ratio (340 nm:380 nm). Cells challengedwith UTP (which increases [Ca²⁺]_(i) in non-transfected cells) caused a[Ca²⁺]_(i) response indicating the presence of a functional G-proteinCa²⁺ linked pathway. Results plotted in FIG. 3 indicate that cellsexpressing wild type NK-1 receptor responded to substance P over a0.01-1000 nM range. Cells expressing wild type anti-sense and variantNK-1 receptor failed to invoke a Ca²⁺ response with substance Pchallenge of 1 nM.

The effect of treatment with varying concentrations of substance-P orwith UTP (3μM) for a 1 min time period on the average change in 340:380ratio levels of COS 7 cultures expressing either wild type or modifiedNK-1 receptors (FIG. 3, n>50 cells for each treatment group (or on thepercentage of COS cells demonstrating a measurable response (Xb) isshown in FIG. 3).

1. A mutant tachykinin receptor in which the three amino acids of theDRY sequence that occurs adjacent to the junction of the TM3 domain with5 intracellular loop 2 are replaced with amino acids whose side chainsare neither lipophilic nor contain charged groups, said receptorexhibiting similar ligand binding characteristics to the wild typereceptor but exhibiting substantially no intra-cellular coupling of thereceptor to the G-protein, whereby there is substantially notransduction of ligand binding 10 signals to the cell; or a fragment ofsaid receptor containing said modified DRY sequence; or an isolatedprotein or polypeptide containing an amino acid sequence at least 80%identical to the above sequence; or a variant thereof with sequentialamino acid deletions from either the C terminus or the N-terminus; or anallelic variant, heterospecific homologue or biologically activeproteolytic or other fragment thereof containing said modified DRYsequence.
 2. A non-human mammalian receptor according to claim
 1. 3. Arat or mouse receptor according to claim
 1. 4. A human receptoraccording to claim
 1. 5. A mutant NIL-1 receptor according to claim 1.6. A mutant NK-2 receptor according to claim
 1. 7. A mutant NK-3receptor according to claim
 1. 8. A receptor according to claim 1,wherein the replacement amino acids are selected from G and A.
 9. Thereceptor of claim 8, wherein DRY is replaced by GGA.
 10. A tachykininreceptor of SEQ ID N^(o)5, or a receptor having at least 80% amino acididentity with the receptor of SEQ ID N^(o)5, and that is capable ofbinding to substance P but is substantially incapable of initiating itsendogenous signal, or a fragment of said receptor.
 11. An isolated cellmembrane incorporating a tachykinin receptor as defined in claim
 1. 12.Any of the following: (a) an isolated nucleic acid molecule comprising apolynucleotide that encodes a tachykinin receptor as claimed in claim 1;(b) an isolated nucleic acid molecule comprising a sequence that ishybridizable to the above sequence; (c) a gene which is the result ofextending the above sequence or any sequence that is hybridizable to theabove sequence; (d) a sequence or gene that is functionally equivalentto the above sequence or to a gene that is an extension of the abovesequence, i.e. that is not identical to the sequence or gene referred tobut functions biologically as equivalent to the sequence or genereferred to, including any allelic variants and heterospecific mammalianhomologues, including artificial or recombinant sequences created fromcDNA or genomic DNA; (e) a recombinant vector comprising the above genesequence; and (f) a host cell transformed with the vector.
 13. Any ofthe following: (a) an isolated nucleic acid molecule having thenucleotide sequence of SEQ ID N^(o)6; (b) an isolated nucleic acidmolecule comprising a sequence that is hybridizable to the abovesequence; (c) a gene which is the result of extending the above sequenceor any sequence that is hybridizable to the above sequence; (d) asequence or gene that is functionally equivalent to the above sequenceor to a gene that is an extension of the above sequence, i.e. that isnot identical to the sequence or gene referred to but functionsbiologically as equivalent to the sequence or gene referred to,including any allelic variants and heterospecific mammalian homologues,including artificial or recombinant sequences created from cDNA orgenomic DNA; (e) a recombinant vector comprising the above genesequence; and (f) a host cell transformed with the vector.
 14. A methodfor producing a receptor protein having an amino acid sequenceas definedin claim 1, which method comprises the steps of: (a) inserting saidnucleic acid sequence into an appropriate vector; (b) culturing, in an aculture medium, a host cell previously transformed or transfected withthe recombinant vector of step (a); (c) harvesting cells containing thereceptor protein obtained from step (b); and (d) separating orpurifying, from said culture medium or from said host cell, thethus-produced receptor protein.
 15. A pharmaceutical compositioncomprising an effective amount of a modified tachykinin ligand asdefined in claim 1 or a nucleic acid sequence encoding said ligand and apharmaceutically and pharmacologically acceptable carrier.
 16. Use of amodified tachykinin receptor as defined in claim 1 in the preparation ofa medicament for the treatment or prophylaxis of a condition associatedwith substance P or other tachykinin (neurokinin) receptor-bindingligand;
 17. A method for the treatment or prevention of a conditionassociated with over-expression or inappropriate expression of anendogenous tachykinin ligand, which method comprises administration to apatient in need thereof of a non-toxic, effective amount of such amodified tachykinin ligand as defined in claim
 1. 18. A method forscreening for therapeutically active compounds, said method comprisingthe following steps: (a) providing a cell line expressing a modifiedtachykinin receptor as defined in claim 1; (b) adding test sample to asolution containing labeled tachykinin ligand and the cell line fromstep (a); (c) incubating the cell line, test sample and labeled ligandmixture from step (b) to allow binding of said ligand and test sample tothe modified tachykinin receptor; (d) optionally, separating thenon-bound labeled ligand from the labeled ligand bound to the modifiedtachykinin receptor; and, if desired, (e) measuring the amount oflabeled ligand that is bound to the modified tachykinin receptor. 19.Use of a modified tachykinin receptor as defined in claim 1 as asubstitute in an assay to identify and/or evaluate entities that bind tothe wild type tachykinin receptor.
 20. Use of a modified tachykininreceptor as defined in claim 1 as a substitute in an assay in order todetermine the concentration of ligand in body fluids in patients witharthritis, pain, migraine, anxiety, schizophrenia, asthma, rheumatoidarthritis, and in gastrointestinal disorders and diseases of the GItract.
 21. An assay procedure comprising the following steps: (a)providing a cell line is provided that expresses a modified tachykininreceptor as defined in claim 1; (b) labeling the cell line; (c) addingthe test sample and labeled cells to a matrix binding SP or otherligand; (d) incubating the labeled cells, test sample and matrix-boundSP or other ligand to allow binding of SP or other ligand and testsample to the expressed modified tachykinin receptor; (e) separating thelabelled non-bound cells from the SP or other ligand bound cells; and,if desired, (f) measuring the amount of labelled cells containing themodified tachykinin receptor that has bound to SF or other ligand. 22.Use of a modified tachykinin receptor as defined in claim 1 in proteintherapy to reduce the effects of an excess of or inappropriatelyproduced endogenous ligand.
 23. A method for treatment of a patient inneed thereof, which comprises administering to said patient acomposition in the form of an aerosol that comprises a modifiedtachykinin receptor as defined in claim
 1. 24. A method for gene therapytreatment of a patient in need thereof, which comprises administering tosaid patient a nucleic acid sequence, virus or plasmid encoding amodified tachykinin receptor as defined in claim 1.